Prolong diamond antifade super resolution imaging mountant

Immunofluorescence microscopy was performed as previously described (Huang et al., 2016 (link)). Briefly, cells were rinsed with phosphate buffered saline (PBS), fixed with 4% (w/v) paraformaldehyde (PFA) for 15 min, and quenched with 50 mM NH₄Cl for 10 min, followed by permeabilization with 0.2% (v/v) Triton X-100 in PBS for 10 min. Cells were then blocked with 1% (w/v) BSA Fraction V (Dot Scientific, DSA30075-100) for 1 h and incubated sequentially with a primary antibody and FITC- or TRITC-labeled secondary antibody diluted in 1% BSA in PBS (PBSB). DNA was stained with 1 μg/mL Hoechst 33342 (ThermoFisher). Coverslips were mounted on glass slides with Moviol and images were taken with a Zeiss Observer Z1 epifluorescence microscope with a 63× oil lens. For super-resolution microscopy, samples were prepared as previously described (Ireland et al., 2020 (link)). Briefly, Alexa Fluor 488-labeled secondary antibodies (ThermoFisher) were used. After washing, coverslips were mounted using ProLong Diamond antifade super-resolution imaging mountant (ThermoFisher). Super-resolution images were taken with a 100× oil lens on a Leica (Wetzlar, Germany) TCS SP8 STimulated Emission Depletion (STED) super-resolution microscope. Images were processed using the NIH ImageJ software. Brightness and contrast were adjusted linearly across all samples to clearly show the Golgi structure.